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Microglia in mouse retina contralateral to experimental glaucoma exhibit multiple signs of activation in all retinal layers.

Rojas B, Gallego BI, Ramírez AI, Salazar JJ, de Hoz R, Valiente-Soriano FJ, Avilés-Trigueros M, Villegas-Perez MP, Vidal-Sanz M, Triviño A, Ramírez JM - J Neuroinflammation (2014)

Bottom Line: In comparison with the control group, a significant increase in the microglial number in the PL, OS, and in the area occupied by Iba-1+ cells in the NFL-GCL, and significant reduction of the arbor area in the PL.Such activation extended beyond the GCL, involving all retinal layers.Differences between the two eyes could help to elucidate glaucoma pathophysiology.

View Article: PubMed Central - HTML - PubMed

Affiliation: Instituto de Investigaciones Oftalmológicas Ramón Castroviejo, Facultad de Medicina, Pab VI, 4a, Avenida Complutense s/n, Universidad Complutense de Madrid, 28040 Madrid, Spain. ramirezs@med.ucm.es.

ABSTRACT

Background: Glaucomatous optic neuropathy, a leading cause of blindness, can progress despite control of intraocular pressure - currently the main risk factor and target for treatment. Glaucoma progression shares mechanisms with neurodegenerative disease, including microglia activation. In the present model of ocular hypertension (OHT), we have recently described morphological signs of retinal microglia activation and MHC-II upregulation in both the untreated contralateral eyes and OHT eyes. By using immunostaining, we sought to analyze and quantify additional signs of microglia activation and differences depending on the retinal layer.

Methods: Two groups of adult Swiss mice were used: age-matched control (naïve, n = 12), and lasered (n = 12). In the lasered animals, both OHT eyes and contralateral eyes were analyzed. Retinal whole-mounts were immunostained with antibodies against Iba-1, MHC-II, CD68, CD86, and Ym1. The Iba-1+ cell number in the plexiform layers (PL) and the photoreceptor outer segment (OS), Iba-1+ arbor area in the PL, and area of the retina occupied by Iba-1+ cells in the nerve fiber layer-ganglion cell layer (NFL-GCL) were quantified.

Results: The main findings in contralateral eyes and OHT eyes were: i) ameboid microglia in the NFL-GCL and OS; ii) the retraction of processes in all retinal layers; iii) a higher level of branching in PL and in the OS; iv) soma displacement to the nearest cell layers in the PL and OS; v) the reorientation of processes in the OS; vi) MHC-II upregulation in all retinal layers; vii) increased CD68 immunostaining; and viii) CD86 immunolabeling in ameboid cells. In comparison with the control group, a significant increase in the microglial number in the PL, OS, and in the area occupied by Iba-1+ cells in the NFL-GCL, and significant reduction of the arbor area in the PL. In addition, rounded Iba-1+ CD86+ cells in the NFL-GCL, OS and Ym1+ cells, and rod-like microglia in the NFL-GCL were restricted to OHT eyes.

Conclusions: Several quantitative and qualitative signs of microglia activation are detected both in the contralateral and OHT eyes. Such activation extended beyond the GCL, involving all retinal layers. Differences between the two eyes could help to elucidate glaucoma pathophysiology.

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Related in: MedlinePlus

CD68 expression in the NFL-GCL after 15 days of unilateral laser-induced OHT. Double immunostaining: Iba-1/CD68. Retinal whole-mount. In contralateral eyes (A, B) CD68 immunoreaction was detected only in some ameboid Iba-1+ cells which exhibited a patchy staining pattern (blank arrowhead). In the retinal whole-mount of OHT eyes (C-H), the greater CD68 immunoreaction was observed in this retinal layer. This was because, in addition to the CD68 immunoreactivity of ameboid Iba-1+ cells (blank arrowhead in C, D), rounded Iba-1+ cells (arrow in C-F) and some rod-like cells (blank arrow in G, H) had intense CD68+ cytoplasmic staining. Rounded Iba-1+ CD68+ cells were adjacent to the retinal vessels (C, D, F), being located mainly close to the optic disc (E) and in the periphery of the retina. In some instances, the processes of ramified Iba-1+ cell surrounded the rounded Iba-1+ cells (arrowhead in C) (NFL-GCL: nerve fiber layer-ganglion cell layer; OD: optic disc; OHT: ocular hypertension; v: retinal blood vessel).
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Figure 5: CD68 expression in the NFL-GCL after 15 days of unilateral laser-induced OHT. Double immunostaining: Iba-1/CD68. Retinal whole-mount. In contralateral eyes (A, B) CD68 immunoreaction was detected only in some ameboid Iba-1+ cells which exhibited a patchy staining pattern (blank arrowhead). In the retinal whole-mount of OHT eyes (C-H), the greater CD68 immunoreaction was observed in this retinal layer. This was because, in addition to the CD68 immunoreactivity of ameboid Iba-1+ cells (blank arrowhead in C, D), rounded Iba-1+ cells (arrow in C-F) and some rod-like cells (blank arrow in G, H) had intense CD68+ cytoplasmic staining. Rounded Iba-1+ CD68+ cells were adjacent to the retinal vessels (C, D, F), being located mainly close to the optic disc (E) and in the periphery of the retina. In some instances, the processes of ramified Iba-1+ cell surrounded the rounded Iba-1+ cells (arrowhead in C) (NFL-GCL: nerve fiber layer-ganglion cell layer; OD: optic disc; OHT: ocular hypertension; v: retinal blood vessel).

Mentions: In naïve eyes, two morphological types of Iba-1+ cells were observed in the NFL-GCL: ramified (Figures 3A, 4A,B) and perivascular (Figures 3A, 4B). In both instances, they are related to the blood vessels. Most Iba-1+ cells had a ramified morphology, varicose processes, and somas located on the retinal vessels and in the intervascular space. From both primary and secondary processes sprouted thin processes that on occasions ended as bulbous-tips (Figures 3A, 4A). Ramified Iba-1+ cells ran parallel to the retinal surface and in some instances, processes penetrating the layer perpendicularly were observed. Perivascular Iba-1+ cells showed elongated morphology and thick somas and processes. These cells were found in the vessel walls (Figures 3A, 4B), specifically on the surface of the retinal large vessels, in the vicinity of the optic nerve, and in the collecting tube venule of the peripheral retina.In contralateral eyes and OHT eyes the two morphological types of Iba-1+ cells described above showed a retraction of the cellular processes in comparison with naïve eyes (Figures 3B,C, 4C-H). In addition, scarce Iba-1+ cells with an ameboid morphology were observed in this layer in contralateral eyes (Figures 3B, 4D inset) and were more frequently found in OHT eyes (Figures 3C, 4G). Notably, two additional morphological types of Iba-1+ cells were observed only in OHT eyes: i) rounded cells (Figures 3C, 4G) and ii) rod-like cells (Figures 3C, 4H). Rounded cells were found adjacent to the major retinal vessels (Figure 5F) or in the neural parenchyma and were observed mainly in the vicinity of the optic disk (Figure 5E) and in the periphery of the retina. In some instances, the processes of ramified Iba-1+ cells surrounded the rounded Iba-1+ cells (Figure 5C). These cells showed a less intense Iba-1+ immunolabeling than did ameboid cells (Figure 4G). Rod-like cells had elongated cell bodies and two processes prominently projected from each pole which were aligned end-to-end, coupling to form trains associated with axons and not related to retinal vessels (Figure 4H).


Microglia in mouse retina contralateral to experimental glaucoma exhibit multiple signs of activation in all retinal layers.

Rojas B, Gallego BI, Ramírez AI, Salazar JJ, de Hoz R, Valiente-Soriano FJ, Avilés-Trigueros M, Villegas-Perez MP, Vidal-Sanz M, Triviño A, Ramírez JM - J Neuroinflammation (2014)

CD68 expression in the NFL-GCL after 15 days of unilateral laser-induced OHT. Double immunostaining: Iba-1/CD68. Retinal whole-mount. In contralateral eyes (A, B) CD68 immunoreaction was detected only in some ameboid Iba-1+ cells which exhibited a patchy staining pattern (blank arrowhead). In the retinal whole-mount of OHT eyes (C-H), the greater CD68 immunoreaction was observed in this retinal layer. This was because, in addition to the CD68 immunoreactivity of ameboid Iba-1+ cells (blank arrowhead in C, D), rounded Iba-1+ cells (arrow in C-F) and some rod-like cells (blank arrow in G, H) had intense CD68+ cytoplasmic staining. Rounded Iba-1+ CD68+ cells were adjacent to the retinal vessels (C, D, F), being located mainly close to the optic disc (E) and in the periphery of the retina. In some instances, the processes of ramified Iba-1+ cell surrounded the rounded Iba-1+ cells (arrowhead in C) (NFL-GCL: nerve fiber layer-ganglion cell layer; OD: optic disc; OHT: ocular hypertension; v: retinal blood vessel).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4128533&req=5

Figure 5: CD68 expression in the NFL-GCL after 15 days of unilateral laser-induced OHT. Double immunostaining: Iba-1/CD68. Retinal whole-mount. In contralateral eyes (A, B) CD68 immunoreaction was detected only in some ameboid Iba-1+ cells which exhibited a patchy staining pattern (blank arrowhead). In the retinal whole-mount of OHT eyes (C-H), the greater CD68 immunoreaction was observed in this retinal layer. This was because, in addition to the CD68 immunoreactivity of ameboid Iba-1+ cells (blank arrowhead in C, D), rounded Iba-1+ cells (arrow in C-F) and some rod-like cells (blank arrow in G, H) had intense CD68+ cytoplasmic staining. Rounded Iba-1+ CD68+ cells were adjacent to the retinal vessels (C, D, F), being located mainly close to the optic disc (E) and in the periphery of the retina. In some instances, the processes of ramified Iba-1+ cell surrounded the rounded Iba-1+ cells (arrowhead in C) (NFL-GCL: nerve fiber layer-ganglion cell layer; OD: optic disc; OHT: ocular hypertension; v: retinal blood vessel).
Mentions: In naïve eyes, two morphological types of Iba-1+ cells were observed in the NFL-GCL: ramified (Figures 3A, 4A,B) and perivascular (Figures 3A, 4B). In both instances, they are related to the blood vessels. Most Iba-1+ cells had a ramified morphology, varicose processes, and somas located on the retinal vessels and in the intervascular space. From both primary and secondary processes sprouted thin processes that on occasions ended as bulbous-tips (Figures 3A, 4A). Ramified Iba-1+ cells ran parallel to the retinal surface and in some instances, processes penetrating the layer perpendicularly were observed. Perivascular Iba-1+ cells showed elongated morphology and thick somas and processes. These cells were found in the vessel walls (Figures 3A, 4B), specifically on the surface of the retinal large vessels, in the vicinity of the optic nerve, and in the collecting tube venule of the peripheral retina.In contralateral eyes and OHT eyes the two morphological types of Iba-1+ cells described above showed a retraction of the cellular processes in comparison with naïve eyes (Figures 3B,C, 4C-H). In addition, scarce Iba-1+ cells with an ameboid morphology were observed in this layer in contralateral eyes (Figures 3B, 4D inset) and were more frequently found in OHT eyes (Figures 3C, 4G). Notably, two additional morphological types of Iba-1+ cells were observed only in OHT eyes: i) rounded cells (Figures 3C, 4G) and ii) rod-like cells (Figures 3C, 4H). Rounded cells were found adjacent to the major retinal vessels (Figure 5F) or in the neural parenchyma and were observed mainly in the vicinity of the optic disk (Figure 5E) and in the periphery of the retina. In some instances, the processes of ramified Iba-1+ cells surrounded the rounded Iba-1+ cells (Figure 5C). These cells showed a less intense Iba-1+ immunolabeling than did ameboid cells (Figure 4G). Rod-like cells had elongated cell bodies and two processes prominently projected from each pole which were aligned end-to-end, coupling to form trains associated with axons and not related to retinal vessels (Figure 4H).

Bottom Line: In comparison with the control group, a significant increase in the microglial number in the PL, OS, and in the area occupied by Iba-1+ cells in the NFL-GCL, and significant reduction of the arbor area in the PL.Such activation extended beyond the GCL, involving all retinal layers.Differences between the two eyes could help to elucidate glaucoma pathophysiology.

View Article: PubMed Central - HTML - PubMed

Affiliation: Instituto de Investigaciones Oftalmológicas Ramón Castroviejo, Facultad de Medicina, Pab VI, 4a, Avenida Complutense s/n, Universidad Complutense de Madrid, 28040 Madrid, Spain. ramirezs@med.ucm.es.

ABSTRACT

Background: Glaucomatous optic neuropathy, a leading cause of blindness, can progress despite control of intraocular pressure - currently the main risk factor and target for treatment. Glaucoma progression shares mechanisms with neurodegenerative disease, including microglia activation. In the present model of ocular hypertension (OHT), we have recently described morphological signs of retinal microglia activation and MHC-II upregulation in both the untreated contralateral eyes and OHT eyes. By using immunostaining, we sought to analyze and quantify additional signs of microglia activation and differences depending on the retinal layer.

Methods: Two groups of adult Swiss mice were used: age-matched control (naïve, n = 12), and lasered (n = 12). In the lasered animals, both OHT eyes and contralateral eyes were analyzed. Retinal whole-mounts were immunostained with antibodies against Iba-1, MHC-II, CD68, CD86, and Ym1. The Iba-1+ cell number in the plexiform layers (PL) and the photoreceptor outer segment (OS), Iba-1+ arbor area in the PL, and area of the retina occupied by Iba-1+ cells in the nerve fiber layer-ganglion cell layer (NFL-GCL) were quantified.

Results: The main findings in contralateral eyes and OHT eyes were: i) ameboid microglia in the NFL-GCL and OS; ii) the retraction of processes in all retinal layers; iii) a higher level of branching in PL and in the OS; iv) soma displacement to the nearest cell layers in the PL and OS; v) the reorientation of processes in the OS; vi) MHC-II upregulation in all retinal layers; vii) increased CD68 immunostaining; and viii) CD86 immunolabeling in ameboid cells. In comparison with the control group, a significant increase in the microglial number in the PL, OS, and in the area occupied by Iba-1+ cells in the NFL-GCL, and significant reduction of the arbor area in the PL. In addition, rounded Iba-1+ CD86+ cells in the NFL-GCL, OS and Ym1+ cells, and rod-like microglia in the NFL-GCL were restricted to OHT eyes.

Conclusions: Several quantitative and qualitative signs of microglia activation are detected both in the contralateral and OHT eyes. Such activation extended beyond the GCL, involving all retinal layers. Differences between the two eyes could help to elucidate glaucoma pathophysiology.

Show MeSH
Related in: MedlinePlus